Isolation and Characterization of Chloroplast DNA from the Marine Chromophyte, Olisthodiscus luteus: Electron Microscopic Visualization of Isomeric Molecular Forms

Abstract

Chloroplast DNA (ctDNA) from the marine chromophytic alga, Olisthodiscus luteus, has been isolated using a whole cel lysis method folowed by CsCI-Hoechst 33258 dye gradient centrifugation. This DNA, which has a buoyant density of 1.691 grams per cubic centimeter was identified as plastidic in origin by enrichment experiments. Inclusion of the nuclease inhibitor aurintricarboxylic acid in all lysis buffers was mandatory for isolation of high molecular weight DNA. Long linear molecules (40 to 48 micrometers) with considerable internal organization comprised the majority of the ctDNA isolated, whereas supertwisted ctDNA and open circular molecules averaging 46 micrometers were occasionally present. Also observed in this study were folded ctDNA molecules with electron dense centers ("rosettes") and plastid DNA molecules which have a tightly wound "key-ring" center. The ctDNA of Olisthodiscus has a contour length that is median to the size range reported for chiorophytic plants.A minor component of the total celular DNA, which originates from a DNase insensitive cellular structure, has a buoyant density of 1.694 grams per cubic centimeter. This DNA consists predominantly oflinear molecules, but open circles 11.5 micrometers in length and rare 22-micrometer dimers were also present.This study represents the first analysis of the extranuclear DNA of a chromophytic alga.

Isolation, physical map and gene map of mitochondrial DNA from the cryptomonad Pyrenomonas salina

“…Similar results were obtained for another species of the Vovocales, Pandorina morum, which was shown to possess a linear mitochondrial genome of about 20 kb [33]. For the chromophyte Olisthodiscus luteus a mitochondrial genome of 50 kb with circular shape has been demonstrated [ 1 ]. The data estimated for P. salina m t D N A match well with those found for other algae, with the exception of the Volvocales.…”

Inverted repeat of Olisthodiscus luteus chloroplast DNA contains genes for both subunits of ribulose-1,5-bisphosphate carboxylase and the 32,000-dalton Q B protein: Phylogenetic implications

“…These mapping data and electron microscopic analysis (16) (37) found that the SS of RbuP2Case is synthesized in vitro from non-poly(A) RNA in the true rhodophytes, Cyanidium caldarum and Porphyridium cruentum. These results indirectly suggest a plastid origin for the rbcS gene in these plants.…”

“…An abstract by Linne von Berg and coworkers (13) The only extensive analysis of any nonchlorophytic plant ctDNA has been done using the alga Olisthodiscus luteus. Restriction analysis (15), contour length measurement (16), and reassociation kinetic analysis (17) …”

Minicircular plastid DNA in the dinoflagellate Amphidinium operculatum

“…Preparative Hoechst 33258-CsCl density centrifugation was done essentially as described by Aldrich and Cattolico (1981). Thus, for H33258-CsCl density gradients, CsCl was added to the DNA suspensions to give a density of 1.69 g/cm 3 .…”